Method for activating a nucleic acid for a polymerase reaction

ABSTRACT

The present invention concerns a method for activating a nucleic acid for a polymerase reaction with the steps: (a) Heating a nucleic acid to a temperature of 55° C. to 80° C., (b) cooling the nucleic acid to a temperature at which a polymerase shows no substantial decrease in activity, and (c) starting the polymerase reaction by the addition of a heat-labile polymerase to the nucleic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a National Phase Application of InternationalApplication No. PCT/EP2006/066223, filed Sep. 11, 2006, which claimspriority to European Patent Application No. 05019665.8 filed Sep. 9,2005, which applications are incorporated herein fully by thisreference.

The present invention concerns a method for activating a nucleic acid,in particular a deoxyribonucleic acid (DNA), for a polymerase reaction,in particular a strand displacement reaction.

By a polymerase reaction, in the sense of the invention, is understoodthe polymerase activity of a nucleic acid polymerase, so thepolymerisation of nucleotides at a free 3′-OH end whereby thecomplementary strand serves as a template. A strand lying possibly 3′from the free 3′-OH-end can thereby either be displaced (stranddisplacement reaction, see below), or this can also be cleaved by a5′-3′ exonuclease activity of the polymerase, and replaced by the newlysynthesised strand (for example in a nick displacement reaction).

A strand displacement reaction (SDR) is a method by which a polymerasereaction with oligonucleotides can be started, whereby during thereaction a peeling off of the “old” strand (“strand displacement”) of adouble-stranded nucleic acid can be affected by the other “old”(complementary) strand, in order to enable a bonding of oligonucleotidesto the other “old” strand. What is important for the reaction is theinitiation, whereby it is possible to differentiate various techniques:

-   (A) A separation of the two hybridised nucleic acid strands (e.g.    DNA strands) can take place through heat denaturing at 95° C. At    these temperatures the two DNA strands are demonstrably separated    from each other, so that oligonucleotides can bond to the denatured    (e.g. separated from each other) DNA strands. The initiation of the    SDR can then take place (e.g. protocol of the GenomiPhi-Kit,    Amersham Biosciences GmbH, Freiburg i. Br., Germany). This method    nevertheless has a substantial disadvantage: Heating to 95° C. leads    to damage to the DNA, e.g. by depurination or strand breakage    ((Suzuki T., Ohsumi S., Makino, K. (1994), Mechanistic studies on    depurination and apurinic site chain breakage in    oligodeoxyribonucleotides, Nucleic Acid Res. 22(23): 4997-5003).-   (B) A separation of the two hybridised nucleic acid strands (DNA    strands) can also take place through an alkali denaturation    (Protocol of the REPLI-g Kit, QIAGEN GmbH, Hilden, Germany).    However, this has the disadvantage, that after the addition of    alkali it has to be neutralised. This means additional pipetting    steps and a change in the reaction environment.-   (C) The next method does not try to achieve strand separation, but    rather it makes use of endonucleases to insert individual strand    breakages, at the 3′-OH end of which the polymerase reaction can    start (cf. e.g. U.S. Pat. No. 6,884,586).-   (d) Finally, a fourth method does not make use of denaturation, as    for example is described by Notomi T., Okayama H., Masubuchi H.,    Yonekawa T., Watanabe K., Amino N., Hase T. (2000), Loop-mediated    isothermal amplification of DNA, Nucleic Acids Res. 15; 28(12):E63.    However, this leads to clearly worse results, as presumably the    individual strand breakages contained in the DNA are used for the    elongation in an SDR.

The task of the present invention is therefore to specify a method forthe activation of a nucleic acid, in particular a deoxyribonucleic acid(DNA), for a strand displacement reaction, which does not feature thedisadvantages of the prior art described above. The inventionaccomplishes this task through a method for activating a nucleic acidfor a polymerase reaction with the steps:

-   -   (a) Heating a nucleic acid to a temperature of 55° C. to 80° C.,    -   (b) Cooling the nucleic acid to a temperature at which a        polymerase shows no substantial decrease in activity, and    -   (c) Starting the polymerase reaction by the addition of a        polymerase to the nucleic acid.

The polymerase being used can be heat-labile or heat-stable. On using aheat-stable polymerase, the method according to the invention foractivating a nucleic acid for a polymerase reaction can alternativelyinclude the step:

-   -   (a) Heating the nucleic acid together with a heat-stable        polymerase to a temperature of 55° C. to 80° C.

Further advantageous embodiments of the present invention are given inthe claims, the description, the examples and the drawing.

In what follows, some of the terms used will be better described.

Strand Displacement Reaction (SDR): Strand displacement reaction is hereunderstood as meaning every reaction in which one polymerase is usedwhich features a strand displacement activity, or in which a reactioncondition is used which makes strand displacement possible. Examples ofthese are strand displacement amplification (SDA), just like multipledisplacement amplification (MDA) or rolling circle amplification (RCA)as well as all subsidiary forms of these reactions, like e.g.restriction-aided RCA (RCA-RCA) or MDA with nested primers, linear andexponential strand displacement reactions or also helicase-dependentamplification (cf. e.g. European patent applications nos. 20050112639,20050074804, 20050069939 and 20050069938, as well as Wang G., Maher E.,Brennan C., Chin L., Leo C., Kaur M., Zhu P., Rook M., Wolfe J. L.,Makrigiorgos G. M. (2004), DNA amplification method tolerant to sampledegradation, Genome Res. November; 14(11):2357-2366; Milla M. A., SpearsP. A., Pearson R. E., Walker G. T. (1998), Use of the restriction enzymeAvaI and exo-Bst polymerase in strand displacement amplification,Biotechniques Mar; 24(3):392-396; Nagamine K., Watanabe K., Ohtsuka K.,Hase T., Notomi T. (2001), Loop-mediated isothermal amplificationreaction using a nondenatured template, Clin Chem. 47(9):1742-1743;Notomi et al 2001 (see above); Lage J. M., Leamon J. H., Pejovic T.,Hamann S., Lacey M., Dillon D., Segraves R., Vossbrinck B., Gonzalez A.,Pinkel D., Albertson D. G., Costa J., Lizardi P. M. (2003), Whole genomeanalysis of genetic alterations in small DNA samples using hyperbranchedstrand displacement amplification and array-CGH, Genome Res.13(2):294-307; and Vincent M., Xu Y., Kong H. (2004), Helicase-dependentisothermal DNA amplification, EMBO Rep. 5(8):795-800).

Strand-Displacement-Polymerase: All polymerases which can carry out astrand displacement are strand displacement polymerases. Examples ofthese are enzymes like phi29-DNA-Polymerase, Cp-1-DNA-Polymerase,PRD1-DNA-Polymerase, phi15-DNA-Polymerase, phi21-DNA-Polymerase,PZE-DNA-Polymerase, PZA-DNA-Polymerase, Nf-DNA-Polymerase,M2Y-DNA-Polymerase, B103-DNA-Polymerase, SF5-DNA-Polymerase,GA-1-DNA-Polymerase, Cp-5-DNA-Polymerase, Cp-7-DNA-Polymerase,PR4-DNA-Polymerase, PR5-DNA-Polymerase, PR722-DNA-Polymerase,L17-DNA-Polymerase, Klenow DNA-Polymerase, Vent DNA Polymerase, DeepVent DNA Polymerase, Bst DNA Polymerase, 9oNm™ DNA Polymerase,Polymerase III-Systeme and Bca DNA Polymerase. The strand displacementpolymerases can also be present in mutated form, e.g. as so-calledexominus variants (i.e. without exonuclease activity).

DNA: Deoxyribonucleic acid (DNA) occurs naturally in organisms, but itcan also occur outside of organisms or could have been added to these.The length of the DNA can differ. DNA can be modified by mutations. TheDNA bases can be modified. The nucleic acid can contain base analogues(e.g. also non-purine or non-pyrimidine analogues) or nucleotideanalogues (e.g. PNA). DNA can contain attachments like e.g. proteins oramino acids.

The present invention therefore concerns a method for activating anucleic acid (in particular a double-stranded DNA) for a stranddisplacement reaction, whereby the method comprises the following steps:(a) Heating the nucleic acid to a (moderate, in comparison to theconventional method) temperature of 55° C. to 80° C.; (b) cooling thenucleic acid to a temperature at which a polymerase shows no substantialdecrease in activity; and (c) starting the strand displacement reactionby the addition of a polymerase to the nucleic acid. The hightemperature of 95° C., applied in a method used until now, which, as isdescribed above, has a negative effect inasmuch as a nucleic acidadopted (e.g. DNA) is substantially damaged by strand breakages anddepurination, can be avoided with the method according to the invention.The separation of double-stranded nucleic acids by means of alkalitreatment with correspondingly disadvantageous attendant circumstancescan also be foregone with the new method. The method according to theinvention therefore offers a possibility to prepare and carry out astrand displacement reaction while conserving the nucleic acid adopted.The method according to the invention is preferably then adopted, if aheat-labile polymerase comes to be used. In the case of the presentinvention, a polymerase is called heat-labile if it only features anactivity of maximum 20% of the initial activity after 10 minutes oftreatment at 65° C., i.e. if the polymerase has been at least 80%inactivated.

The variant described above is preferably then adopted, if a heat-labilepolymerase comes to be used. If, however, a heat-stable polymerase isused, which endures at least a short-term heating up to 80° C.,preferably up to 70° C., and especially preferably up to 65° C. withoutnoteworthy loss of activity, then this can be added already at step (a),and cooling of the reactants before addition of the polymerase can beomitted. The alternative method therefore comprises step (a) heating thenucleic acid to a (moderate, in comparison to the conventional method)temperature of 55° C. to 80° C. In the sense of the present invention,heat-stable polymerases are understood as meaning all non-heat-labilepolymerases.

The moderate temperature, to which the cells or isolated DNA are heated,lies in between 55° C. and 80° C., preferably 60° C. and 70° C. andespecially preferably at 65° C. The heating of the DNA of the cells cantake place directly in the SDR reaction mixture, for example.

The invention describes an activation of a nucleic acid (in particularDNA) for a strand displacement reaction by means of a moderate heatingstep. The method according to the invention correspondingly comprisesthe following partial steps as part of the use of isolated nucleic acid(DNA) and a heat-labile strand displacement polymerase: (1) The nucleicacid (DNA) is heated to a moderately high temperature. (2) The nucleicacid (DNA) is cooled down, whereby the temperature after the coolingprocess may have as its maximum temperature one at which the polymerasedoes not yet clearly lose its activity. The nucleic acid is preferablycooled down to a temperature of 4° C. to 45° C., especially preferablyto a region of 15° C. to 42° C. and really especially preferably to aregion of 25° C. to 37° C. (3) The SDR reaction is started by theaddition of the (heat-labile) polymerase.

The variant described above is preferably then adopted, if a heat-labilepolymerase is used. But should a heat-stable polymerase be adopted, thenthis can already be added at step (1).

The method according to the invention can be used not only for pure orpurified DNA, but also for DNA which is still contained in a cellularbond. The method for the use of DNA which is still contained in thecellular bond, and of a heat-labile strand displacement polymerase,correspondingly has the following partial steps: (1) The nucleic acid(DNA) is heated to a moderately high temperature. (2) The cellscontaining DNA are cooled down, whereby the temperature after thecooling process may have as its maximum temperature one at which thepolymerase does not yet clearly lose its activity. (3) The SDR reactionis started by the addition of polymerase.

In both methods the heat-labile strand displacement polymerase can alsobe replaced by a heat-stable strand displacement polymerase. Then thepartial step (1) can be carried out directly with the polymerase.

The diagrams show:

FIG. 1 The yield of the reactions from example 1;

FIG. 2 The Ct values of the real-time PCR from example 1;

FIG. 3 The yield of the reactions from example 2;

FIG. 4 The Ct values of the real-time PCR from example 2;

FIG. 5 The Ct values of the real-time PCR from example 3;

FIG. 6 The yield of the reactions from example 4;

FIG. 7 The Ct values of the real-time PCR from example 4;

FIG. 8 The Ct values of the real-time PCR from example 5;

FIG. 9 The Ct values of the real-time PCR of the locus 11/12 fromexample 6;

FIG. 10 The Ct values of the real-time PCR of the locus 665 from example6.

The invention is described more closely in the following by means ofexamples.

EXAMPLE 1

The example should show that by a simple temperature activation step anSDR (here a multiple displacement amplification, MDA) starting withwhole blood is made possible, which as regards the DNA yield and the DNAquality is comparable to a reaction according to the prior art (controlreaction).

Reaction according to the invention: the MDA reaction was carried outwith the REPLI-g reagents (QIAGEN GmbH, Hilden, Germany). 0.5, 1 and 2μl of whole blood as appropriate, stabilised by EDTA or citrate, werebrought up to a volume of 39.5 μl with 12.5 μl 4× reaction mix (thiscontains oligonucleotides with a random sequence, reaction buffersolution and dNTPs) and water, and subsequently heated to 65° C. Aftercooling down to room temperature (around 20-25° C.), the DNA polymerasefrom the REPLI-g kit was added. The reaction was then carried out at 30°C. for 6 h.

Control reaction according to the prior art: the MDA reaction wascarried out with the REPLI-g reagents (QIAGEN): 0.5 μl whole blood,stabilised by EDTA, was displaced with 2.5 μl phosphate-buffered saltsolution (PBS, from the REPLI-g Kit from QIAGEN). Subsequently, 3.5 μlof the freshly-added denaturation buffer solution (360 mM KOH, 9 mMEDTA, 100 mM DTT) was added to it and incubated on ice for 10 min. Themixture was neutralised after incubation with solution B (REPLI-g kit).The mixture was brought up to a volume of 49.5 μl with 12.5 μl 4×reaction mix from the REPLI-g kit (this contains oligonucleotides with arandom sequence, reaction buffer solution and dNTPs) and distilledwater. After that, 0.5 μl of the DNA polymerase from the REPLI-g kit wasadded. The reaction was then carried out at 30° C. for 6 h.

After the end of the reaction, the DNA concentration was measured withPicoGreen according to the manufacturer's protocol (Molecular ProbesInc., Eugene, Oreg., USA). 10 ng of the MDA-DNA was inserted for areal-time PCR (polymerase chain reaction). 4 different loci wasinvestigated for their representation in the amplificat:

(a) a sequence which was named Sat (Primer sequences:

(SEQ ID NO: 1)and (SEQ ID NO: 2) Sat1.1 TCTTTCCACTCCATTGCAT(b) a sequence from the B-actin gene

(SEQ ID NO: 3) Primer 1 GTCTCAAGTCAGTGTACAGG (SEQ ID NO: 4)Primer 2 GTGATAGCATTGCTTTCGTG(c) a sequence which emanates from the locus named “1004” (Test: (SEQ IDNO: 5) Primer 1: (SEQ ID NO: 6) Primer 2: (SEQ ID NO: 7) and(d) a sequence which emanates from the locus named “699”

(SEQ ID NO: 8) (Test: TGAACTGCTCCTTGGCAGGGATTT, (SEQ ID NO: 9)Primer 1: TGCTCCCTGTCCCATCTG, (SEQ ID NO: 10)Primer 2: AGACAGTATGCCTTTATTTCACCC).

The real-time PCR reactions were carried out in the QuantiTect MasterMix (QIAGEN) according to the protocol instructions.

The representation of the loci in the amplified DNA was measured in Ctvalues (threshold cycles=Ct). The Ct value is the PCR cycle in thereal-time PCR, by which the fluorescence signal can be measured for thefirst time. The relative frequency of a sequence in the test cantherefore be ascertained by the Ct value. If, for example, a Ct value is1 cycle smaller than in a comparison test, then this value correspondsto an initial quantity of DNA in the test run approximately 2-timeshigher than in the control run for the sequence measured.

The result of example 1 can be summarised as follows: (1) The yield fromthe reactions in which the DNA was activated according to the inventionby a 65° C. step is comparable to the yield from the control reaction.(2) The representation of the sequences in the amplified DNA is alsocomparable, if 0.5 μl of blood is added. Solely the sequence of the Satlocus is lower than in the control reaction. (3) Larger volumes than 0.5μl have an inhibiting effect on the MDA reaction with 65° C. activation.This can be seen from the higher Ct values (i.e. worse representation ofthe sequences in the amplified DNA).

The yield of the reactions from example 1 is depicted graphically inFIG. 1. FIG. 2 show the Ct values of the real-time PCR from example 1.

EXAMPLE 2

This example serves to show that too low temperatures during atemperature activation step can impair the quality of the DNA whicharises during the SDR.

Reaction according to the invention: the MDA reaction was carried outwith the REPLI-g reagents (QIAGEN): 0.5 μl whole blood, stabilised byEDTA, was brought up to a volume of 39.5 μl with 12.5 μl 4× reaction mix(this contains oligonucleotides with a random sequence, reaction buffersolution and dNTPs) and water and activated by various temperatures (30,40, 45, 50, 55, 60 and 65° C.). After cooling down to room temperature(around 20-25° C.), the DNA polymerase from the REPLI-g kit was added.The reaction was carried out at 30° C. for 6 h.

Control reaction according to the prior art: the MDA reaction wascarried out with the REPLI-g reagents (QIAGEN): 0.5 μl whole blood,stabilised by EDTA, was displaced with 2.5 μl PBS (REPLI-g kit).Subsequently, 3.5 μl of the freshly-added denaturation buffer solution(360 mM KOH, 9 mM EDTA, 100 mM DTT) was added to it and incubated on icefor 10 min. The mixture was neutralised after incubation with solution B(REPLI-g kit). The mixture was brought up to a volume of 49.5 μl with12.5 μl 4× reaction mix from the REPLI-g kit (this containsoligonucleotides with a random sequence, reaction buffer solution anddNTPs) and distilled water. After that, 0.5 μl of the DNA polymerasefrom the REPLI-g kit was added. The reaction was carried out at 30° C.for 6 h.

After the end of the reaction, the DNA concentration was measured withPicoGreen according to the manufacturer's protocol (Molecular Probes).10 ng of the MDA-DNA were added for a real-time PCR. 2 different lociwere investigated for their representation in the amplificat:

(a) a sequence which emanates from the locus named “1004”

(SEQ ID NO: 5) (Test: TGATGGCATTACTGGCACTTTGAGTTTTAC, (SEQ ID NO: 6)Primer 1: GTCTTTAGCTGCTGAGGAAATG and (SEQ ID NO: 7)Primer 2: AGCAGAATTCTGCACATGACG), as well as(b) a sequence which emanates from the locus named “699”

(SEQ ID NO: 8) (Test: TGAACTGCTCCTTGGCAGGGATTT, (SEQ ID NO: 9)Primer 1: TGCTCCCTGTCCCATCTG, (SEQ ID NO: 10)Primer 2: AGACAGTATGCCTTTATTTCACCC).

The real-time PCR reactions were carried out in the QuantiTect MasterMix (QIAGEN) according to the protocol instructions.

The representation of the loci in the amplified DNA was measured in Ctvalues (threshold cycles=Ct). The Ct value is the PCR cycle in thereal-time PCR, by which the fluorescence signal can be measured for thefirst time. The relative frequency of a sequence in the test cantherefore be ascertained by the Ct value. If, for example, a Ct value is1 cycle smaller than in a comparison test, then this value correspondsto an initial quantity of DNA in the test run approximately 2-timeshigher than in the control run for the sequence measured.

The result of example 2 can be summarised as follows: (1) The yield fromthe reactions in which the DNA was activated according to the inventionby a 30 to 65° C. step is comparable to the control reaction. (2) Therepresentation of the sequences in the amplified DNA is also onlysomewhat worse than in the control reaction, if the activation iscarried out at 60 or 65° C. With an activation of the DNA for the stranddisplacement reaction under 60° C., the representation of the observedsequences becomes worse.

The yield of the reactions from example 2 is depicted graphically inFIG. 3. FIG. 4 shows the Ct values of the real-time PCR from example 2.

EXAMPLE 3

In should be shown in this example that within specific temperatureboundaries a simple temperature activation before the SDR does notaffect the quality of the DNA arising in the SDR in comparison to areaction according to the prior art (control reaction).

Reaction according to the invention: the MDA reaction was carried outwith the REPLI-g reagents (QIAGEN). 0.5 μl whole blood, stabilised byEDTA, was brought up to a volume of 39.5 μl with 12.5 μl 4× reaction mix(this contains oligonucleotides with a random sequence, reaction buffersolution and dNTPs) and water, and activated by various temperatures(60, 65, and 70° C.). After cooling the reaction composition down toroom temperature (around 20-25° C.), the DNA polymerase from the REPLI-gkit was added. The reaction was carried out at 30° C. for 6 h.

Control reaction according to the prior art: the MDA reaction wascarried out with the REPLI-g reagents (QIAGEN): 0.5 μl whole blood,stabilised by EDTA, was displaced with 2.5 μl PBS (REPLI-g kit).Subsequently, 3.5 l of the freshly-added denaturation buffer solution(360 mM KOH, 9 mM EDTA, 100 mM DTT) was added to it and incubated on icefor 10 min. The mixture was neutralised after incubation with solution B(REPLI-g kit). The mixture was brought up to a volume of 49.5 μl with12.5 μl 4× reaction mix from the REPLI-g kit (this containsoligonucleotides with a random sequence, reaction buffer solution anddNTPs) and with distilled water. After that, 0.5 μl of the DNApolymerase from the REPLI-g kit was added. The reaction was carried outat 30° C. for 6 h.

After the end of the reaction, the DNA concentration was measured withPicoGreen according to the manufacturer's protocol (Molecular Probes).10 ng of the MDA-DNA were added for a real-time PCR. 4 different lociwere investigated for their representation in the amplificat:

(a) a sequence which emanates from the locus named “1004”

(SEQ ID NO: 5) (Test: TGATGGCATTACTGGCACTTTGAGTTTTAC, (SEQ ID NO: 6)Primer 1: GTCTTTAGCTGCTGAGGAAATG, (SEQ ID NO: 7)Primer 2: AGCAGAATTCTGCACATGACG) and(b) a sequence which emanates from the locus named “699”

(SEQ ID NO: 8) (Test: TGAACTGCTCCTTGGCAGGGATTT, (SEQ ID NO: 9)Primer 1: TGCTCCCTGTCCCATCTG, (SEQ ID NO: 10)Primer 2: AGACAGTATGCCTTTATTTCACCC).c) a sequence which was named Sat

(Primer sequences: (SEQ ID NO: 1) Sat1.1 TCTTTCCACTCCATTGCAT and(SEQ ID NO: 2) Sat1.2 GGAATGGAATCAACCCAA(d) a sequence from the B-actin gene

(SEQ ID NO: 3) Primer 1 GTCTCAAGTCAGTGTACAGG (SEQ ID NO: 4)Primer 2 GTGATAGCATTGCTTTCGTG

The real-time PCR reactions were carried out in the QuantiTect MasterMix (QIAGEN) according to the protocol instructions.

The representation of the loci in the amplified DNA was measured in Ctvalues (threshold cycles=Ct). The Ct value is the PCR cycle in thereal-time PCR, by which the fluorescence signal can be measured for thefirst time. The relative frequency of a sequence in the test cantherefore be ascertained by the Ct value. If, for example, a Ct value is1 cycle smaller than in a comparison test, then this value correspondsto an initial quantity of DNA in the test run approximately 2-timeshigher than in the control run for the sequence measured.

The result can be summarised as follows. The representation of thesequences in the amplified DNA is partially better in the preparationaccording to the invention than in the control reaction, if theactivation is carried out at 60° C., 65° C. or 70° C.

FIG. 5 shows the Ct values of the real-time PCR analysis of DNA from theSDR reaction from example 3.

EXAMPLE 4

The example serves to show that by a simple temperature activation stepan SDR (here a multiple displacement amplification, MDA) starting withisolated genomic DNA is made possible, which as regards DNA yield andDNA quality is comparable to a reaction according to the prior art(control reaction).

Reaction according to the invention: the MDA reaction was carried outwith the REPLI-g reagents (QIAGEN). 2.5 μl of a solution of genomic DNAfrom human cells (concentration: 4 ng/μl) was brought up to a volume of39.5 μl with 12.5 μl 4× reaction mix (this contains oligonucleotideswith a random sequence, reaction buffer solution and dNTPs) and water,and activated by an incubation step at 65° C. After cooling the reactioncomposition down to room temperature (around 20-25° C.), the DNApolymerase from the REPLI-g kit was added. The reaction was carried outat 30° C. for 6 h.

Control reaction according to the prior art: the MDA reaction wascarried out with the REPLI-g reagents (QIAGEN). 2.5 μl genomic DNA fromhuman cells (concentration: 4 ng/μl) were displaced by 2.5 μlfreshly-added denaturation buffer solution (50 mM KOH, 1.25 mM EDTA) andincubated for 3 min at room temperature. The mixture was neutralisedafter incubation with a 1:10 dilution of solution B (REPLI-g kit). Themixture was brought up to a volume of 49.5 μl with 12.5 μl 4× reactionmix from the REPLI-g kit (this contains oligonucleotides with a randomsequence, reaction buffer solution and dNTPs) and distilled water. Afterthat, 0.5 μl of the DNA polymerase from the REPLI-g kit was added. Thereaction was carried out at 30° C. for 6 h.

After the end of the reaction, the DNA concentration was measured withPicoGreen according to the manufacturer's protocol (Molecular Probes).10 ng of the MDA-DNA was added for a real-time PCR. 1 locus wasinvestigated for representation in the amplificat:

(a) a sequence which emanates from the locus named “1004”

(SEQ ID NO: 5) (Test: TGATGGCATTACTGGCACTTTGAGTTTTAC, (SEQ ID NO: 6)Primer 1: GTCTTTAGCTGCTGAGGAAATG, (SEQ ID NO: 7)Primer 2: AGCAGAATTCTGCACATGACG).

The real-time PCR reactions were carried out in the QuantiTect MasterMix (QIAGEN) according to the protocol instructions.

The representation of the loci in the amplified DNA was measured in Ctvalues (threshold cycles=Ct). The Ct value is the PCR cycle in thereal-time PCR, by which the fluorescence signal can be measured for thefirst time. The relative frequency of a sequence in the test cantherefore be ascertained by the Ct value. If, for example, a Ct value is1 cycle smaller than in a comparison test, then this value correspondsto an initial quantity of DNA in the test run approximately 2-timeshigher than in the control run for the sequence measured.

The result was that the yield from the reactions in which the DNA wasactivated by a 65° C. step, was almost twice as high as in the controlaccording to the prior art. The representation of the sequences in theamplified DNA is comparable in the test according to the invention andin the control test.

The yield of the reactions from example 4 is depicted graphically inFIG. 6. FIG. 7 shows the Ct values of the real-time PCR from example 4.

EXAMPLE 5

In this example it is shown that by a simple denaturation step atincreasingly high temperatures, specific sequences can no longer beamplified in an SDR (here a multiple displacement reaction).

Test reactions: the MDA reaction was carried out with the REPLI-greagents (QIAGEN): 10 ng isolated DNA was incubated at temperatures of75° C., 85° C. or 95° C. for 10 min. Alternatively, an alkalidenaturation was carried out in a KOH buffer solution in controlreactions. After the chemical denaturation with KOH, the solution wasneutralised, in order not to influence the MDA reaction conditions.Subsequent to the heat treatment or chemical denaturation, the solutioncontaining DNA was brought up to a volume of 39.5 μl with 12.5 μl 4×reaction mix (this contains oligonucleotides with a random sequence,reaction buffer solution and dNTPs) and water. After that, the DNApolymerase from the REPLI-g kit was added to the reaction mixtures. Thereaction was carried out at 30° C. for 6 h.

After the end of the reaction, the DNA concentration was measured withPicoGreen according to the manufacturer's protocol (Molecular Probes).10 ng of the MDA-DNA was added for a real-time PCR. 2 different lociwere investigated for their representation in the amplificat:

(a) a sequence which emanates from the locus named 1004

(SEQ ID NO: 5) (Test: TGATGGCATTACTGGCACTTTGAGTTTTAC, (SEQ ID NO: 6)Primer 1: GTCTTTAGCTGCTGAGGAAATG, (SEQ ID NO: 7)Primer 2: AGCAGAATTCTGCACATGACG) and(b) sequence which emanates from the locus named 699

(SEQ ID NO: 8) (Test: TGAACTGCTCCTTGGCAGGGATTT, (SEQ ID NO: 9)Primer 1: TGCTCCCTGTCCCATCTG, (SEQ ID NO: 10)Primer 2: AGACAGTATGCCTTTATTTCACCC).

The real-time PCR reactions were carried out in the QuantiTect MasterMix (QIAGEN) according to the protocol instructions.

The representation of the loci in the amplified DNA was measured in Ctvalues (threshold cycles=Ct). The Ct value is the PCR cycle in thereal-time PCR, by which the fluorescence signal can be measured for thefirst time. The relative frequency of a sequence in the test can betherefore be ascertained by the Ct value. If, for example, a Ct value is1 cycle smaller than in a comparison test, then this value correspondsto an initial quantity of DNA in the test run approximately 2-timeshigher as compared to the control run for the sequence measured.

Result:

-   -   (1) With increasing temperature in the heat treatment of DNA,        increasingly high CT values are measured.    -   (2) Both loci behave differently upon heat treatment: In the        cases of locus 699, a Ct shift of 9.6 cycles could be measured,        if you compare the values with a treatment at 75° C. or 95° C.        In any case, in the cases of locus 1004 another Ct shift of 5        cycles is measured. I.e. with increasing temperature of the heat        treatment of the DNA, the loci measured in the MDA amplification        product were proving worse and worse, even though introduced        into a unified quantity of the amplification product (10 ng) for        real-time PCR.

A Ct shift of 9.6 or 5 cycles corresponds to a representation of thesequence 699 or 1004 respectively 780* or 30* times less in theamplification product of the DNA, which was heated to 95° C. beforeamplification, compared to a DNA treated at 75° C. (* these values arebased on the fact that in every cycle of the real-time PCR a duplicationtakes place).

FIG. 8 shows the Ct values of the real-time PCR from example 5.

EXAMPLE 6

This example shows at which temperatures an optimal temperatureactivation step takes place, in order to achieve as good a sequencerepresentation in an SDR (here a multiple displacement amplification) aspossible.

Test reactions: the MDA reaction was carried out with the REPLI-greagents (QIAGEN): 10 ng isolated DNA was incubated at varioustemperatures for 5 min. Alternatively, DNA was prepared in controlreactions in a multiple-step method consisting of (1) addition of KOH,(2) incubation of the DNA in the KOH solution for 5 min and (3)neutralisation of the alkaline solution for the REPLI-g reaction.

Subsequent to the heat treatment or chemical denaturation, the solutioncontaining DNA was amplified in a REPLI-g reaction. The reaction wascarried out at 33° C. for 8 h.

After the end of the reaction, the DNA concentration was measured withPicoGreen according to the manufacturer's protocol (Molecular Probes).10 ng of the MDA-DNA was added for a real-time PCR. 2 different lociwere investigated for their representation in the amplificat:

(a) a sequence which emanates from the locus named 11/12

(SEQ ID NO: 11) (Primer 1: TTTCTGTAACAGCTAAGGAC, (SEQ ID NO: 12)Primer 2: TAGGGTGCTTAGCTGTTAAC) and(b) sequence which emanates from the locus named 665

(SEQ ID NO: 13) (Primer 1: CTCTTGCTCAGCCTATATAC, (SEQ ID NO: 14)Primer 2: GTAGAAAATGTAGCCCATTAC.

The real-time PCR reactions were carried out in the QuantiTect MasterMix (QIAGEN) according to the protocol instructions.

The representation of the loci in the amplified DNA was measured in Ctvalues (threshold cycles=Ct). The Ct value is the PCR cycle in thereal-time PCR, by which the fluorescence signal can be measured for thefirst time. The relative frequency of a sequence in the test can betherefore be ascertained by the Ct value. If, for example, a Ct value is1 cycle smaller than in a comparison test, then this value correspondsto an initial quantity of DNA in the test run approximately 2-timeshigher than in the control run for the sequence measured.

Result:

(1) With increasing temperature in the heat treatment of DNA,increasingly high CT values are measured.

(2) The lowest CT value (so the best representation of the lociinvestigated here) emerges with a thermal preparation at a temperatureof 65° C. to 85° C.

(3) At these loci better Ct values were measured at temperatures of65-85° C. than with the reference treatment by means of KOH.

(4) It can be deduced from the comparison example 5, in which worse CTvalues were already measured at 85° C. than in the reference treatmentby means of KOH, that the optimal treatment temperature is dependent onthe locus of the genome in a cell.

FIG. 9 shows the Ct values of the real-time PCR of the locus 11/12 fromexample 6. FIG. 10 shows the Ct values of the real-time PCR of the locus665 from example 6.

The invention claimed is:
 1. A method for activating a non-damagednucleic acid for a strand displacement reaction with the steps: (a)heating a reaction mixture comprising a non-damaged nucleic acid to atemperature between 55° C. to 80° C., (b) cooling the reaction mixtureto a temperature of 4° C. to 45° C., and (c) adding a polymerase to thecooled reaction mixture of step (b), wherein the polymerase added instep (c) starts the strand displacement reaction, wherein the reactionmixture is not an alkali solution, and wherein two or more primers areadded prior to or subsequent to step (a).
 2. The method of claim 1,wherein the polymerase added in step (c) is a heat-labile polymerase. 3.The method of claim 1, wherein the polymerase added in step (c) is aheat-stable polymerase.
 4. The method of claim 1, wherein the stranddisplacement reaction is a multiple displacement reaction.
 5. The methodof claim 1, wherein the nucleic acid is a DNA.
 6. The method of claim 1,wherein the reaction mixture in step (a) is heated to a temperature of60° C. to 70° C.
 7. The method of claim 6, wherein the reaction mixturein step (a) is heated to a temperature of 65° C.
 8. The method of claim1, wherein the reaction mixture in step (b) is cooled down to atemperature of 15° C. to 42° C.
 9. The method of claim 8, wherein thereaction mixture in step (b) is cooled down to a temperature of 25° C.to 37° C.
 10. The method of claim 1, wherein the reaction mixture ofstep (a) comprises purified nucleic acid in an aqueous solution.
 11. Themethod of claim 1, wherein the reaction mixture of step (a) comprisesnucleic acid in a cell.
 12. The method of claim 1, wherein the two ormore primers are oligonucleotides with a random sequence.
 13. The methodof claim 1, wherein the polymerase is phi29-DNA-Polymerase.
 14. A methodfor activating a non-damaged nucleic acid for a strand displacementreaction with the steps: (a) heating a reaction mixture comprising anon-damaged nucleic acid to a temperature between 55° C. to 65° C., (b)cooling the reaction mixture to a temperature of 4° C. to 45° C., and(c) adding a polymerase to the cooled reaction mixture of step (b),wherein the polymerase added in step (c) starts the strand displacementreaction, and wherein two or more primers are added prior to orsubsequent to step (a).